Synthetic oligonucleotides have been an integral part of scientific research for the last twenty-five years, but initial methods of oligonucleotide synthesis were very labor-intensive. Organic synthesis on solid supports was pioneered by Merrifield, R. B. (See Journal of the American Chemical Society, 85:2149, for protein synthesis on solid supports). Advances in automation of oligonucleotide synthesis began in the early 1980's when reaction columns containing solid support were employed (Bender et al., U.S. Pat. No. 4,353,989). Oligonucleotide synthesizers were developed that added the necessary reagents to several reaction columns simultaneously. The synthesis of a typical 20-mer oligonucleotide can take several hours, and the first generation synthesizers typically ran no more than four columns. High throughput synthesis required either a great deal of time or a significant amount of synthesizers. When using these synthesizers, a technician will place a known amount of a support derivatized with the corresponding base into a column that will be used for oligomer synthesis.
Currently, methods have advanced to enable a technician to load multi-well plates wherein each well operates as a reaction column (McGraw et al., U.S. Pat. No. 5,368,823). With the automated synthesizers such as described in McGraw, a technician manually loads each of the 96 wells with one of the four synthesis membrane supports that correspond to the four possible bases that are pre-attached to the support.
The multi-well plate synthesizer is a major advancement in reducing the amount of instrumentation required for high throughput synthesis, and they also can potentially minimize the amount of reagents used in the synthesis of oligonucleotides. The reagents used are expensive, and the loss of reagent through waste or error can significantly increase the cost of the synthesis of an oligonucleotide. Any methods that would minimize the amount of reagents required to be present in a given well at a given cycle and/or used in multiple cycles would enhance the synthesizer's performance and minimize cost. Additionally, the removal of any procedures in the current state of the art that increase the likelihood of error in the synthesis would be advantageous. These steps include the loading of the derivatized supports to the respective column. As mentioned above, there are typically four different derivatized supports for each DNA or RNA synthesis that correspond to the four different deoxy- or ribonucleotide bases respectively. For either DNA or RNA synthesis, the four different types of supports are visually indistinguishable, and a support for RNA synthesis would be indistinguishable from a support for DNA synthesis. Any errors in the placement of the supports into the column will not be noticed until after the synthesis is complete.
To support oligonucleotide synthesis, either high throughput such as with 96 or 384-well plates or in smaller-scale throughput, methods and compositions are needed that reduce the likelihood of errors in oligonucleotide synthesis.
The proposed invention provides compositions and methods that reduce the likelihood of error during organic synthesis on solid supports, including oligonucleotide synthesis. The invention also provides a novel high-throughput oligonucleotide synthesizing format with color-coded supports that provide a more efficient and an error-free method of loading the initial base-containing supports into the synthesis wells of plates or individual columns. The invention also provides a method for verification of the loaded wells. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.